Prokaryotic Promoters and Transcription

Qualitative PCR only determines if a sequence is present
Quantitative PCR determines if a sequence is present and the amount
Regular/Traditional PCR is qualitative
Advantages: fast, easy, sensitive, can amplify small amounts from degraded/fixed samples
Disadvantages: often requires prior knowledge of the sequence, short products, infidelity of replication
Components needed for a PCR reaction and their roles:
- Template DNA: blueprint for amplification
- Primers: dictate where to amplify
- DNA polymerase (Taq): synthesizes new strands, heat-resistant
- Nucleotides (dNTPs): building blocks of DNA
PCR cycle steps and temperatures:
- Denaturation (95°C): separates DNA strands
- Annealing (~55°C): primers bind to target sequence
- Extension (72°C): new DNA synthesized

Components needed for PCR reaction and their role:
- Template DNA: The DNA sequence to be copied
- Primers: Short DNA sequences that define the region to be amplified
- DNA Polymerase: An enzyme that synthesizes new DNA strands by adding nucleotides to the 3' end of the primer. Taq polymerase is commonly used due to its heat stability.
- Nucleotides (dNTPs): Deoxyribonucleoside triphosphates, the building blocks of DNA
PCR cycle steps and temperatures:
- Denaturation: Heating the DNA to a high temperature (typically 94-98°C) to separate the double-stranded DNA into single strands.
- Annealing: Lowering the temperature (typically 50-65°C) to allow the primers to bind to their complementary sequences on the single-stranded DNA.
- Extension: Raising the temperature again (typically 72°C) to allow the DNA polymerase to synthesize new DNA strands by extending the primers.
Why Taq Polymerase is used:
- Heat stable, can function at high temperatures required for PCR.
Why PCR amplification is exponential
- Each DNA molecule produced can be used as a template for further replication.

Length: Usually 20 nucleotides
Avoid these when designing primers:
- Tandem repeats
- Secondary structures (hairpins)
- 3' end complementarity

Binding Protein Hot Start Method: Binding proteins bind to primers, then denaturation step inactivates them.
Antibody Hot Start Method: Polymerase is bound by antibody preventing it from working until denaturation.

RT-PCR converts RNA into cDNA using reverse transcriptase, then amplifies cDNA using PCR
cDNA is single-stranded DNA produced from an RNA template using reverse transcriptase

Quantitative PCR
Similar to regular PCR but measures the amplified DNA in real time
Advantages: Monitor amplification real-time and accurate quantification of starting DNA material.
Disadvantages: More expensive, requires more extensive instrumentation.

Ct value: Cycle number when fluorescence crosses a set threshold. Lower Ct values mean higher starting amounts of DNA target.
Standard curve method: Use Ct to determine the starting DNA amount for unknown samples.
AACt method: Used to compare Ct values of samples to a control (normalized)

Used to determine the initial starting amount of target template in experimental samples
Slope measures reaction efficiency.
Requires a dilution series of known template concentrations

First Generation - Sanger Sequencing:
- Based on chain termination, low cost, high accuracy
Second Generation (NGS) - NGS:
- High throughput, lower accuracy, cost-effective
Third Generation - Long-Read Sequencing:
- Very long reads, relatively lower accuracy but cost-effective for particular applications

Amplifies DNA molecules in micro-droplets.
Ensures each droplet contains only one DNA molecule, increasing specificity of reaction.
Useful for high-throughput applications, minimizes contamination

More sensitive than qPCR
Distributes template DNA into numerous droplets
Measures DNA in individual droplets to determine concentration with high accuracy.

DNA polymerase synthesizes new DNA strands
Chain terminator nucleotides (ddNTPs) create different lengths, fragments read on a gel.

Ion Torrent PGM: • Measures hydrogen ion released when nucleotides are added to the growing DNA strand.
Molecular Beacons, TaqMan Probes: • Use fluorescent probes to detect DNA synthesis.
Oxford Nanopore: • DNA strands pass through a nanopore, changes in current are detected to determine nucleotide sequence.

Combining short DNA fragments (reads) into longer, contiguous sequences (contigs) with overlapping regions
Challenges: repetitive sequences, errors in sequencing, and the need to use reference genes.

Transcription: Making mRNA from DNA
Translation: Making protein from mRNA
Operon: cluster of genes transcribed as a single mRNA in prokaryotes
Promoter: DNA region where RNA polymerase binds to initiate transcription
Leader Sequences: Region in some operons preceding the transcribed protein-encoding sequence, important in transcriptional regulation.
Repressors: Proteins that bind to DNA sequences (operator) to block RNA polymerase, hindering transcription.
Activators: Proteins that bind to DNA to stimulate RNA polymerase activity.
Attenuation: a secondary regulatory mechanism of some operons, in which termination of transcript occurs early in some conditions.
Constitutive: Always transcribed with or without a regulatory protein
Inducible: Transcribed only when needed based on the environmental signals for an induced response
Repressible: Transcribed unless blocked by a regulatory protein

mRNA (messenger RNA): codes for proteins
tRNA (transfer RNA): carries amino acids to ribosomes
rRNA (ribosomal RNA): forms part of ribosomes
ncRNA (non-coding RNA): various functions, many of which are regulatory (e.g., sRNA).